Oncoinhibin

ABSTRACT

The present invention provides a novel human cytokine termed Oncoinhibin. The protein Oncoinhibin is secreted by human erythroblastoid cells, has a molecular weight of approximately 28 kDa and exerts diverse neoplastic activity. The present invention also provides a method for the preparation of human Oncoinhibin.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to cytokines. More specifically,the present invention relates to a novel cytokine with broadanti-neoplastic activity.

2. Description of the Related Art

Cell growth appears to be regulated by a balance between growthstimulatory and growth inhibitory molecules. An imbalance in thesegrowth regulatory cytokines has been proposed as one of the mechanismsof tumor growth.

Several cytokines which stimulate the growth of tumor and normal cellshave been described. These include, e.g., epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet derived growth factor (PDGF),insulin-like growth factors (IGF), interleukins (IL), colony stimulatingfactors (CSF) and transforming growth factors (TGF-α and TGF-β).

In contrast, other cytokines selectively inhibit the growth of certaintumor cells. These include, e.g., interferons (IFN), lymphotoxin (LT),tumor necrosis factor (TNF), oncostatin M, amphiregulin, interleukin-1(IL-1), interleukin-6 (IL-6) and TGF-β.

These growth stimulatory and growth inhibitory cytokines can bedifferentiated from each other based on their source, their specificityagainst tumor targets, their physio-chemical properties and theirprimary structure. Thus, the identification and characterization ofgrowth regulatory cytokines is of critical importance in theunderstanding of cellular growth, including growth of neoplasms.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a novelcomposition of matter. This novel composition of matter, termedOncoinhibin, is secreted by human erythroblastoid cells, has a molecularweight of approximately 28 kDa on SDS-PAGE and exhibits diverseanti-neoplastic activity.

In one embodiment the present invention provides a method for preparinga novel human cytokine termed Oncoinhibin. The method comprisesincubating human erythroblastoid cells, inducing the production ofOncoinhibin and harvesting conditioned cell supernatants.

In yet another embodiment of the present invention, there is provided amethod of purifying human Oncoinhibin. This method comprises the stepsof ultrafiltering conditioned cells supernatants containing humanOncoinhibin. Subsequently in human Oncoinhibin and dialyzing theultrafiltered supernatants. Subseqneutly DEAE Affigel bluechromotography, Sodium dodecyl Sulfate-polyacrylamide gelelectrophoresis and reverse phase high performance liquid chromatographyis performed to purify human Oncoinhibin.

In another embodiment of the present invention, there is provided anovel immunomodulator for activating lymphocytes, monocytes andneutrophils to kill tumor cells. The novel immunomodulator compriseshuman Oncoinhibin. Also provided is a novel growth factor forstimulating the growth of normal cells. This growth factor compriseshuman Oncoinhibin.

In other embodiments of the present invention there are providedpharmaceutical compositions of Oncoinhibin and methods of treating aneoplastic cell comprising the administration of an effective dose ofOncoinhibin.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of this specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting of their scope. In the invention made of equally effectiveequivalent embodiments.

FIG. 1 illustrates that K-562 cell conditioned supernatants inhibit thegrowth of MCF-7 cells.

FIG. 2 depicts the standard bioassay for Oncoinhibin.

FIG. 3 shows the production of Oncoinhibin by K-562 cells in thepresence and absence of serum.

FIG. 4 depicts the effects of phorbol ester on the induction ofOncoinhibin.

FIG. 5 shows the effects of ultrafiltration on Oncoinhibin activity.

FIG. 6 shows the characterization of Oncoinhibin by gel permeationchromatography.

FIG. 7 shows the elution of Oncoinhibin activity from SDS polyacrylamidegel electrophoresis.

FIG. 8 depicts the SDS-PAGE analysis of Oncoinhibin.

FIG. 9 shows the binding elution of Oncoinhibin activity from DEAEAffigel blue (upper panel) and from a Q-sepharose column (lower panel).

FIG. 10 illustrates the dose dependent anti-proliferative effects ofOncoinhibin and TNF.

FIG. 11 shows dose dependent proliferative effects of Oncoinhibin onnormal human foreskin fibroblasts.

FIG. 12 shows the effect of Oncoinhibin on actinomycin D treated murineL-929 cells.

FIG. 13 shows a northern blot analysis for TNF and LT of K-562 cellsuntreated and treated with phorbol esters.

FIG. 14 shows the comparison of growth inhibitory effects of Oncoinhibin(panel A) and oncostatin M (panel B) on human melanoma A375 cells.

FIG. 15 shows the growth inhibitory effects of oncostatin M and IL-6 onnormal fibroblasts.

FIG. 16 shows the effect of IL-6 on human breast tumor (MCF-7) cells.

FIG. 17 depicts the effects of antibodies against interferon-γ on theOncoinhibin activity on human breast tumor cells (MCF-7).

FIG. 18 shows the inhibitory effects of interferon-γ on TNF (upperpanel) but not Oncoinhibin (lower panel) on human foreskin fibroblasts.

FIG. 19 shows the growth rate of human breast tumor MCF-7 cells in theabsence and presence of Oncoinhibin.

FIG. 20 shows the effect of time of exposure of MCF-7 cells toOncoinhibin.

FIG. 21 shows comparison of the effect of Oncoinhibin with TNF on themorphology of human breast tumor MCF-7 cells.

DETAILED DESCRIPTION OF THE INVENTION

Various cell lines including mouse connective tissue cell line L929 (CCL1), K-562 (CCL-243), U-937 (CRL-1543), HL-60 (CCL-240), Raji (CCL-86),Jurkat (CRL-8163), BT 20 (HTB-1 9), MCF-7 (HTB-22), SK-BR-3 (HTB-30),ZR-75-1 (CRL-1500), RPMI 7951 (HTB-66), A375 (CRL-1619), A-431(CRL-1555), ME-1 80 (HTB 83), OVCAR-3 (HTB-161), He La (CCL-2), Hep-2(HB-8065), and NIH 3T3(CRL-1618) were obtained from American TypeCulture Collection, (Rockville, Md.). TNF-resistant NIH 3T3 cells wereisolated as described by K. Totpal, R. LaPushin, H. N. Ananthaswamy andB. B. Aggarwal, Lymphokine and Cytokine Res. 10 (1991) 359-367. Cellswere tested for mycoplasma contamination using the DNA-based assay kitpurchased from Gen-Probe (San Diego, Calif.).

All cell cultures were maintained in continuous exponential growth byweekly passage. Some of the cells were subcultivated twice a week. Cellswere routinely grown in RPMI 1640 medium supplemented with glutamine (2mM), penicillin (100 units/ml), streptomycin (100 μg/ml), and fetalbovine serum (10%) in a humidified incubator in 5% CO₂ in air.

The conditioned supernatants of human erythroblastoid cell line K-562produces an activity which is growth inhibitory to human breast tumorcell line MCF-7. Due to its ability to inhibit the growth of tumor cellsand not that of normal cells, this activity is termed "Oncoinhibin".

For the production and induction of Oncoinhibin, human erythroblastoidcell line K-562 was grown in RPMI 1640 medium containing 10% fetalbovine serum supplemented with glutamine (2 mM), penicillin (100units/ml), and streptomycin (100 μg/ml). Cells were harvested bycentrifugation when a density of 0.8×10⁶ cells/ml was reached, the cellswere washed once with medium without serum and transferred to serum-freeconditions in RPMI-1640 medium containing glutamine, penicillin andstreptomycin. For production of Oncoinhibin, 1×10⁶ /ml of these cellswere incubated for 48 hours in T175 flask (Falcon) under stationaryculture conditions in RPMI 1640 medium without serum and then treatedwith phorbol ester (100 ng/ml) for 48 hours at 37° C. Thereafter, theconditioned cell supernatants were harvested by centrifugation, filteredthrough 0.22 micron filter (Falcon) and stored at 4° C. until furthercharacterization. In order to concentrate the Oncoinhibin conditionedmedia from K-562 cell lines was ultrafiltered by PM-10 membrane (AmiconCorp.) and then dialyzed with 20 mM Tris, pH 8.0.

With reference to FIG. 1, inhibitory activity of Oncoinhibin on tumorcell growth was examined by three separate methods. These methodsincluded (1) counting cells on hemocytometer after trypan blud staining;(2) crystal violet dye-uptake method; and (3) by tritiated thymidineincorporation method. Oncoinhibin clearly inhibits the growth of MCF-7cells by all three methods in a dose dependent manner. Due to theconvenience and sensitivity, MCF-7 cell line was used as a target todevelop the bioassay for Oncoinhibin. The inhibition of tritiatedthymidine incorporation by Oncoinhibin was found to be a highlysensitive method to detect this cytokine.

The bioassay for Oncoinhibin consists of plating 5×10³ cells in 96 wellflat bottom well plates in 0.1 ml of RPMI 1640 medium with 10% FCSovernight at 37° C. in a CO₂ incubator. Then the media is removed, atwo-fold serial dilution of the test sample is added in a total finalvolume of 0.1 ml and incubation is continued for 24 hours at 37° C.During last 6 hours, tritiated thymidine (0.5 μCl/0.05 mi/ well) isadded. At the end of 24 hours incubation period, media is poured-off andcells are detached with 0.1 ml of Trypsin (0.5%) and EDTA (5.3 mM)treatment for 30 minutes at 37° C. Cells are harvested by using PHDCambridge cell harvestor and cell-incorporated radioactivity isdetermined by beta counter. The data was expressed as % relativeviability which is defined as amount of dpm taken up by cells in thepresence of Oncoinhibin divided by the dpm incorporated in the presenceof the media alone, multiplied by 100. The amount of Oncoinhibinrequired to inhibit the viability by 50% was defined as one unit of thecytokine. As is seen in FIG. 1, Oncoinhibin exerts a dose-dependentdependent inhibition of tumor cell growth as illustrated by all threemethods.

With reference to FIG. 2, a clear dose-dependent response by MCF-7 toOncoinhibin could be observed within 24 hours. The reciprocal of thedilution of the sample needed to achieve 50% inhibition in thymidineincorporation was defined as one unit of Oncoinhibin.

With reference to FIG. 3, the production of Oncoinhibin under serum-freeconditions was examined. Serum-free conditions were used due to thedifficulty in purifying the proteins from samples containing serum.These results clearly indicate that the Oncoinhibin is secreted by K-562cells even in the absence of serum.

We examined whether different agents can induce production ofOncoinhibin. Calcium ionophore, Concanavalin A, Phytohemagglutinin, andphorbol ester were examined. FIG. 4 shows that phorbol ester canincrease the production of Oncoinhibin. Thus, phorbal ester can be usedto optimize the production of Oncoinhibin from K-562 cells. Anapproximately four fold increase in the production of Oncoinhibin wasobserved when cells were exposed to phorbol ester (100 ng/ml). Optimuminduction of Oncoinhibin was observed when cells were incubated withphorbol ester for 48 hours (Table I) and at a cell-density of 1×10⁶cells per ml of the media (Table II).

                  TABLE I                                                         ______________________________________                                        Time Course of Induction of Oncoinhibin                                       from Human K-562 Cells by Phorbol Ester                                                    Uninduced                                                                             Induced                                                                 Relative Cell                                                  Time (hrs)     Viability (%)                                                  ______________________________________                                         0             94        --                                                    6             100       60                                                   24             84        33                                                   48             67        16                                                   72             41        40                                                   ______________________________________                                    

K-562 cells (1×10⁶ /ml) were cultured in serum-free media (RPMI-1640)either in the presence or absence of the phorbol ester (100 ng/ml) at37° C. in a CO₂ incubator for different times and then the conditionedmedia was harvested by centrifugation. Samples were tested on MCF-7cells at 1:2 fold-serial dilutions as indicated in Materials andMethods.

                  TABLE II                                                        ______________________________________                                        Optimization of production of Oncoinhibin in presence and                     Absence of phorbol ester at different Cell-Densities*                                       Uninduced                                                                             Induced                                                 Cell Number     Relative Cell                                                 (× 10.sup.6 /ml)                                                                        Viability (%)                                                 ______________________________________                                        0.01            76        83                                                  0.1             89        56                                                  0.5             65        41                                                  1.0             37        26                                                  1.5             50        28                                                  ______________________________________                                         *K-562 cell were incubated at the indicated celldensity in serumfree medi     (RPMI1640) for 72 hrs at 37° C. in a CO.sub.2 incubator. The           conditioned media was harvested by centrifugation and tested at twofold       serial dilution on MCF7 cells as described in Materials and Methods.     

With reference to FIG. 5, to purify and characterize Oncoinhibin, thecell conditioned media as concentrated by ultrafiltration using a 10,000molecular weight cut-off (PM-10) membrane. The activity in the fractionnot retained by the filter (Flow through or FT) was lower whereas thatin the retained fraction or concentrate (C) was proportionately higherthan that of the standard buffer (starting material, SM). The resultsindicate that Oncoinhibin activity is retained and concentrated. Theseresults also indicate a molecular weight higher than 10,000 forOncoinhibin.

In the gel permeation fast protein liquid chromatographic experiment ofFIG. 6, panels a and b, a sample of Oncoinhibin was applied onto aSuperose-6 column (Pharmacia) pre-equilibrated with phosphate-bufferedsaline containing 0.1% bovine serum albumin and 0.01% sodium azide. Thecolumn was run at room temperature with a flow rate of 0.5 ml per minuteand the size of each fraction was 0.5 ml. The column was calibrated withmolecular weight standards (Schwarz/Mann, Cambridge, Mass.). The latterincluded Apoferritin (480 kDa), Alpha Amylase (20 kDa), Gamma-Globulin(160 kDa), Bovine Serum Albumin (67 kDa), Ovalalbumin (45 kDa),Chymotrypsinogen (24 kDa) and Cytochrome C (12.4 kDa).

With reference to FIG. 6, it appears that Oncoinhibin has an approximatemolecular weight of 25 kDa. The molecular weight of Oncoinhibin wasexamined by gel permeation Fast Protein Liquid Chromatography onSuperose-6 column under nondenaturing conditions. The results of gelfiltration run in phosphate buffered saline, pH 7.4 show two major peaksof activity, one coinciding with the excluded volume and the second peakcorresponding to an approximate molecular weight of around 25 kDa (FIG.6, panel B).

In the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) experiments of FIGS. 7 and 8, a 15% polyacrylamide gel wasrun essentially according to U. K. Laemmli (Nature 227 (1970) 680-685,incorporated by reference herein) and proteins were visualized by silverstaining. For preparative gel electrophoresis and elution of activity, aportion of the gel before fixing and staining was sliced with a razorblade into over 40 different slices, eluted in a test tube with 50 mMammonium bicarbonate by diffusion overnight, the fractions were dialyzedagainst 20 mM Tris, pH 8.0 and then assayed for biological activity.

With reference to FIG. 7, to further confirm the molecular weight ofOncoinhibin, SDS-PAGE analysis was performed. After electrophoresis, thegels were sliced, eluted in 50 mM ammonium bicarbonate overnight andassayed for Oncoinhibin activity. Greater than 50% of the appliedOncoinhibin activity was recovered in the molecular weight region ofaround 30 kDa. Less than 10% activity was also found in an area near thedye front.

With reference to FIG. 8, a rerun of the biologically active fraction onSDS-PAGE and silver staining of gels showed a single major band at amolecular weight of around 28 kDa.

With reference to FIG. 9, panels a and b, Oncoinhibin binding to andelution from anion exchange resins was examined. One column (1 cm×5 cm)was packed with an anion exchange resin Q-Sepharose and thenequilibrated with 20 mM Tris, pH 8.0 (equilibration buffer). A sample ofOncoinhibin dialyzed against the equilibration buffer was loaded ontothe column with a flow rate of 0.5 ml/minute. The column was rinsed withthe equilibration buffer and then resolved with a step-up gradients ofNaCl (0-1M). Various fractions were analyzed for protein concentrationand for bioactivity.

DEAE Affigel Blue Chromatography: A second column (1 cm×5 cm) was packedwith DEAE Affigel Blue resin and then equilibrated with 20 mM Tris, pH8.0 (equilibration buffer). A sample of Oncoinhibin pre-equilibratedagainst the equilibration buffer by dialysis was loaded onto the columnwith a flow rate of 0.5 ml/minute. The column was rinsed with theequilibration buffer and then resolved with a step-up gradients of NaCl(0-1M). Various fractions were analyzed for protein concentration andfor bioactivity.

Panels a and b of FIG. 9 show that Oncoinhibin binds and can be elutedfrom anion exchange resins. To both the Q-sepharose and DEAE affigelBlue resins, Oncoinhibin activity bound in 20 mM Tris, pH 8.0 buffer.The bound Oncoinhibin could be eluted with 0.2M NaCl in 20 mM Trisbuffer from DEAE affigel Blue and with 0.5M NaCl from Q-sepharose.

Oncoinhibin inhibits the growth of wide variety of tumor cells. (TableVII) The antiproliferative activity of Oncoinhibin was examined bytritiated thymidine incorporation.

                  TABLE III                                                       ______________________________________                                        Differences in tumor cell                                                     specificity between TNF and Oncoinhibin                                                           Relative Cell                                                                 Viability (%)                                             Cell Lines            TNF    Oncoinhibin                                      ______________________________________                                        Erythroblastoid cell line (K562)                                                                    94     2                                                Histiocytic Lymphoma (U-937)                                                                        54     1                                                Histiocytic Lymphoma (U-937-CF-1)                                                                   13     0                                                Promyelocytic Lymphoma (HL-60)                                                                      100    1                                                Burkitt Lymphoma (Raji)                                                                             76     0                                                T cell Lymphoma (Jurket)                                                                            74     36                                               Myelogenous Leukemia (KG-1)                                                                         43     8                                                Myelogenous Leukemia (KG-1a)                                                                        60     13                                               Myelogenous Leukemia (ML-1a)                                                                        59     0.1                                              Myelogenous Leukemia (ML-1b)                                                                        35     0.1                                              Monocytic Leukemia (THP-1)                                                                          81     1                                                Breast Carcinoma (BT20)                                                                             24     2                                                Breast Carcinoma (BT20TNFR)                                                                         60     3                                                Breast Carcinoma (MCF7)                                                                              1     0                                                Breast Carcinoma (SK-BR3)                                                                           52     0                                                Breast Carcinoma (ZR-75-1)                                                                           6     1                                                Melanoma (RPMI 7951)  73     0                                                Melanoma (A375)       76     0                                                Epidermoid Carcinoma (A-431)                                                                        82     0                                                Cervical Carcinoma (ME-1 80)                                                                        20     0                                                Ovarian Carcinoma (OVCAR-3)                                                                         27     1                                                Cervical Carcinoma (HeLa)                                                                           55     14                                               Hepatoma (HepG-2)     83     20                                               Retinoblastoma (Weri-Rb-1)                                                                          60     33                                               Retinoblastoma (Y-79) 92     46                                               Glioblastoma (LG)     84     2.3                                              Murine Fibroblasts (NIH 3T3)                                                                        11     35                                               Murine Fibroblasts (LTR1 000)                                                                       85     27                                               Murine Fibroblasts (L929)                                                                            0     1                                                Normal Human Foreskin Fibroblasts                                                                   311    189                                              ______________________________________                                         Tumor cells (5000/well) were incubated with TNF (0.2 μg/ml) or             Oncoinhibin (induced by phorbol ester and concentrated) for 72 hours at       37° C. and then relative cell viability (%) was determined by          Thymidine incorporation as described previously.                         

With reference to FIG. 10, panels A-D, the dose response effects ofOncoinhibin on some of the cell lines listed in Table VII were examinedand compared to TNF. It is clear from the results shown in Table VII andFIG. 10 that, besides MCF-7, Oncoinhibin can inhibit the growth ofseveral different types of leukemias, melanomas, carcinomas andhepatomas. The growth of murine cells was also inhibited. Thus, itappears that, unlike interferons, Oncoinhibin is not species-specific.

With reference to FIGS. 11 and 12, for growth assays, cells were platedfor overnight in 0.1 ml of the medium (RPMI-1640 with 10% FBS) in96-well Falcon plates. Thereafter, the medium was removed and a serialdilution of human Oncoinhibin was layered in 0.1 ml of the volume. After72 hours of incubation at 37° C., the medium was removed and viablecells were monitored by crystal violet staining according to theprocedure as described by B. B. Aggarwal, Human lymphotoxin, Meth. ofEnzymol., 116:441-448 (1985), incorporated herein by reference. A dyeuptake method to examine cell viability correlates with cell numberdetermined by detachment with a trypsin solution and microscopiccounting with hemocytometer. The percentage of relative cell viabilitywas calculated as optical density in the presence of the test sampledivided by optical density in the absence of the test sample (medium)multiplied by 100. For LT and TNF, cytotoxicity assays were carried outsimilar to growth inhibition assays except that 20×10³ L-929 cells weretreated with actinomycin D (1 μg/ml) along with the cytokine for 24hours.

Cell Growth-Stimulatory Assays. Cell growth-stimulatory assays werecarried out essentially according to the procedure described by Vilceket al., J. Exp. Med. 163:632-643 (1986), incorporated herein byreference. Briefly, confluent human diploid foreskin fibroblasts atpassage level 12-16 (corresponding to approximately population doublinglevels 24-32) were used for cell growth stimulatory assays. To determinethe effect of human Oncoinhibin, cells (8×10³ /well) were plated in 0.1ml of the medium (RPMI-1640+10% FBS) in 96-well Falcon plates. Afterovernight incubation in a CO₂ incubator at 37° C., the medium wasremoved and a serial dilution of the cytokine was layered in 0.2 ml ofthe volume. After 5 days incubation, media was decanted-off and cellswere stained with crystal violet. All determinations were made intriplicate. Percent relative cell viability was calculated as indicatedfor growth-inhibitory assays.

For [³ H] TdR incorporation assays, human fibroblasts were cultured andtreated with the cytokine for 5 days. During the last 24 hours,tritiated thymidine (6.7 Ci/mmole; New England Nuclear, Boston, Mass.)was added to each well (0.5 μCi/well). Thereafter, the culture mediumwas removed, the wells were washed twice with phosphate-buffered salineand the cells were detached by the addition of a solution of trypsin(0.5%) with EDTA (5.3 mM). The cell suspension was then harvested withthe aid of PHD cell harvestor (Cambridge Technology Inc. Watertown,Mass.) and lysed by washing with distilled water. Radioactivity bound tothe filter was measured in a liquid scintillation counter (Model 1600TR; Packard Co., Meriden, Conn.). Thymidine incorporation in humanfibroblast determined by this method correlates with cell growth. Intumor cell growth inhibition studies, cells were incubated with thecytokine for 3 days in a total of 0.1 ml final volume and then monitoredfor thymidine incorporation.

With reference to FIG. 11, Oncoinhibin appears to stimulate the growthof normal human foreskin fibroblasts. While inhibiting the growth oftumor cells, Oncoinhibin enhanced the proliferation of normal humanforeskin fibroblasts.

With reference to FIG. 12, similar to TNF and LT, Oncoinhibin cytolysesactinomycin D treated L-929 cells. The antitumor activity of Oncoinhibinagainst L-929 cells cannot be neutralized by either anti-LT or anti-TNFantibodies (Table IV). That is, no significant amounts of TNF or LT weredetected by ELISA assay in Oncoinhibin preparations (Table V). Table IIIshows that several tumor cell types (e.g.; SK-BR-3, HeLa, A-431,OVCAR-3, A375, and RPMI-7951) which are resistant to TNF/LT, areequisitely sensitive to Oncoinhibin. Oncoinhibin can also bedistinguished from TNF/LT on the basis of cell lines isolated forresistance to TNF/LT (NIH 3T3-LTR and BT-20 TNFR), were found to besensitive to Oncoinhibin. (Table III)

                  TABLE IV                                                        ______________________________________                                        Lack of Neutralization of Oncoinhibin activity by Monoclonal                  Antibodies Against Tumor Necrosis Factor and Lymphotoxin*                                              Relative Cell                                                                            Neutral-                                  Sample   Antibody        Viablity % ization %                                 ______________________________________                                        Oncoinhibin                                                                            None            58         0                                                  +Anti-TNF       58         0                                                  +Anti-LT        60         0                                                  +Anti-TNF + Anti LT                                                                           56         4                                         TNF      None            39         0                                                  +Anti-TNF       100        100                                                +Anti-LT        44         5                                         LT       None            35         0                                                  +Anti-LT        90         90                                                 +Anti-TNF       38         0                                         ______________________________________                                         *Oncoinhibin, TNF and LT were incubated with either AntiTNF or AntiLT         antibodies at 37° C. for 1 hour and then directly assayed for          remaining nonneutralized TNF or LT activity on Actinomycin D treated L929     cells as described before (4).                                           

                  TABLE V                                                         ______________________________________                                        Determination of Various Cytokines                                            in a crude preparation of Oncoinhibin                                         derived from K-562 cells*                                                     Cytokine       Levels (pg/ml)                                                 ______________________________________                                        TNF-α    3 ± 6                                                       TNF-β (LT)                                                                              2 + 0                                                          IL-1β        0                                                           IL-6           155 ± 7                                                     IL-8           >2000                                                          ______________________________________                                         Serum-free K562 cellconditioned media was used as a source of Oncoinhibin     and the level of various cytokines were determined by standard ELISA          assays (R&D System). ND is not determined                                

TNF and LT are products of monocytes and lymphocytes, respectively, andinhibit the growth of a wide variety of cells. Similar to Oncoinhibin,lymphotoxin and TNF inhibit the growth of MCF-7 cells, but largeconcentrations of TNF and LT (10,000 units/ml) are needed.

To further confirm that Oncoinhibin is neither TNF nor LT, a Northernblot analysis was performed to look for the gene for either of thecytokines. In the northern blot analysis, phorbol ester treated anduntreated K-562 cell cultures seeded at 1×10⁶ cells/ml in 75-cm flaskswere incubated with protein kinase C activator for 24 hours and thenharvested by centrifugation. Total RNA was extracted from cells by theguandinium isothiocyanatephenol-chloroform method shown by Chirgwin, etal., Biochem. 18:5294-5299 (1979) and Maniatis, et al., MolecularCloning 188-209 (1982), incorporated herein by reference. Routinely, RNAwith 260 nm/280 nm absorbance ratio of greater than 1.9 and yield ofapproximately 100 μg RNA per 20×10⁶ cells was obtained.

For electrophoresis, 20 μg of RNA was fractionated on 0.8% agarous gelscontaining 2.2M formaldehyde at 75-100 V for approximately 3 hours.Thereafter, gels were exposed to diethyl pyrocarbonate-treated water at68° C. for 1 hour, and then the RNA transferred to Hybond nylonmembranes (Amersham Corp., Arlington Heights, Ill.). After transfer (3hours), the filter was rinsed twice with SSC (SSC: 0.15M sodiumchloride, 15 mM sodium acetate, 15 mM sodium citrate, pH 7.0) and placedin a microseal bag.

Prehybridization was carried out at 65° C. for 1 hour in a buffercontaining 7% SDS, 500 mM sodium phosphate, 1 mM EDTA, pH 7.2(hydridization buffer). Filters were then hybridized with TNF or LT cDNAprobes (specific activity 2×10⁸ cpm/μg DNA). After hydridization,membranes were washed several times at 65° C. in hydribidzation buffercontaining salmon sperm DNA (200 μg/ml). The filters were exposed toKodak XAR-5 film at -70° C. for 1-3 days. Procedures for sequentialcycles of prehybridization, hybridization, washes and filter strippingwere performed. Equal loading of lanes was demonstrated by examinationof gels after ethidium bromide staining and also by rehybridization ofsame filters with cDNAs for either actin or glyceraldehyde 3-phosphatedehydrogenase (GAPDH). Band densitometry was performed by eitherscanning the filter for radioactive counts with blot analyzer Betascope603 (Betagen Corp., Waltham, Mass.) or by scanning autoradiogram foroptical density by using Scanning Densitometer (Helena Laboratories Inc.Beaumont, Tex.). As shown in FIG. 13, no expression of mRNA for eitherLT or TNF was observed in K-562 cells. Furthermore, gel filtration andSDS-PAGE experiments confirm that the molecular weight of Oncoinhibin isdifferent from that of either TNF or LT.

With reference to FIG. 19, human breast tumor cells grow rapidly inculture in the media. When Oncoinhibin was added to the culture,however, no growth of these cells was observed. In order to determinethe time of exposure needed to inhibit the growth of these cells, thecells were exposed to Oncoinhibin for different times. With reference toFIG. 20, the growth-inhibitory effects of Oncoinhibin are terminatedwhen the cytokine is removed from the media. These results suggest theneed for continuous presence of the Oncoinhibin.

With reference to FIG. 21, the morphology of the MCF-7 cells wasexamined after treatment with Oncoinhibin and compared to TNF. Theresults indicate a difference in the method by which TNF and Oncoinhibininhibit the growth of breast tumor cells. TNF induces rounding-up of thecells which leads to their detachment from the dish, while Oncoinhibininduces enlargement or swelling of cells. The latter may result fromeffects of Oncoinhibin on the permeability of the cells. Oncoinhibinalso inhibits the colony formation of human breast tumor cells.

Oncoinhibin Stabiity Studies. Oncoinhibin was treated with organicsolvent (acetonitrile or methanol or propanol), acidic solvents (HCl,trichloroacetic acid or acetic acid), alkaline solvents (NaOH, Ammoniumhydroxide) or detergents (SDS, Tween 10) for two hours at roomtemperature, then dialyzed against 20 mM Tris-HCl, pH 8.0 overnight in acold room and assayed for biological activity. (Tables VI and VIII).

For thermostability experiments, Oncoinhibin was exposed to differenttemperatures for different times and the biological activity wasdetermined directly. The biological activity of Oncoinhibin was found tobe stable to 80° C. for 60 minutes but approximately 50% loss ofactivity was observed when exposed to 100° C. for 30 minutes (Table VI).

                  TABLE VI                                                        ______________________________________                                        Thermostability of Oncoinhibin at Different Temperatures                      Temperature                                                                              Time       Activity Remaining                                      (°C.)                                                                             (min)      U/Ml          %                                         ______________________________________                                         4         60         256           100                                       80         30         210           82                                                   60         230           90                                        90         30         200           78                                                   60         190           74                                        100        30         128           50                                                   60         110           43                                        ______________________________________                                    

In Table VII, Oncoinhibin was treated with pronase E, trypsin,chymotrypsin and V8 Staph protease and then analyzed for its biologicalactivity. The results indicate that Oncoinhibin activity can becompletely abolished by pronase E, thus suggesting that it is a protein.Its activity was found to be partially sensitive to trypsin treatmentand completely resistant to chymotrypsin and V8 protease.Deoxyribonuclease also has no effect on the activity of Oncoinhibin.

                  TABLE VII                                                       ______________________________________                                        Effect of Proteolytic Enzymes on the activity of Oncoinhibin                  Enzymes      Conc.      Activity Remaining (%)                                ______________________________________                                        None         10% (w/w)  100                                                   Pronase E    10% (w/w)  0.2                                                   Trypsin      10% (w/w)  50                                                    V8 Staph. Protease                                                                         10% (w/w)  100                                                   Chymotrypsin 10% (w/w)  100                                                   Deoxyribonuclease                                                                          10% (w/w)  100                                                   ______________________________________                                    

Oncoinhibin was incubated with various enzymes at 37° C. for 24 hours in20 mM Tris buffer at pH 8.0, then reaction was stopped by addition of10% serum and assayed for Oncoinhibin activity.

Stability of Oncoinhibin to Detergents: A sample of Oncoinhibin wastreated with different concentrations of either SDS, a negativelycharged detergent or Tween 20, a neutral detergent for 2 hours, dialyzedand then assayed for biological activity. Bovine serum albumin treatedwith the same detergent was used as a control. The results of theseexperiments is shown in Table V. No. loss of biological activity wasobserved on treatment of the protein either with SDS or Tween 20. Incase of SDS, it appears that there is a significant increase in thebiological activity of Oncoinhibin in a dose-dependent manner. Theincrease was not significant with Tween 20.

                  TABLE VIII                                                      ______________________________________                                        Effect of Detergent, pH and Organic Solvent                                   Treatment on the Stability of Oncoinhibin                                                            Activity Remaining                                     Agent          Conc.   U/ml           %                                       ______________________________________                                        Detergent Stability:                                                          Sodium dodecyl sulfate                                                                       None    19             100                                                    0.01%   23             124                                                    0.05%   45             243                                                    0.10%   61             330                                                    0.50%   59             319                                     Tween-20       0.01%   24             130                                                    0.05%   24             130                                                    0.10%   16              88                                     pH Stability:                                                                 Glycine   pH 2.0   0.1M    240          300                                   Acetic acid                                                                             pH 2.4   1.0M    256          320                                   Sodium acetate                                                                          pH 4.0   0.1M    83           104                                   Sodium acetate                                                                          pH 6.0   0.1M    76            95                                   Tris-HCL  pH 8.0   0.02M   80           100                                   NaHCO3    pH 10.0  0.1M    92           115                                   NH4OH     pH 11.4  1.0M    185          231                                   Organic Solvent Stability:                                                                           S/P                                                    None           --      42             100                                     Methanol       50%     81/2           198                                     Propanol       50%     56/3           140                                     Acetone        70%     23/26          117                                     Ethanol        70%     56/4           143                                     Acetonitril    50%     153/4          374                                     ______________________________________                                         Oncoinhibin samples were treated at room temperature with the various         agents for 1 hour in 20 mM Tris buffer pH 8.0, dialyzed overnight and the     assayed for biological activity remaining. S and P represents supernatant     and pellet fractions.                                                    

Stability of Oncoinhibin to reducing agents: Oncoinhibin was treatedwith different concentrations of dithiothreitol for 2 hours, thendialyzed and assayed for biological activity. The results of theseexperiments are shown in Table VI. It is clear that the activity ofOncoinhibin is unstable to the treatment of DTT. A 50% loss in activitywith 1 mM DTT and 67% loss with 100 mM DTT was observed.

                  TABLE IX                                                        ______________________________________                                        Stability of Oncoinhibin to Various Treatments                                Agent          Conc.     Activity  Percent                                    ______________________________________                                        Reducing Agents (DTT)                                                                        --        256       100                                                        1 mM     128       50                                                         10 mM     96       38                                                        100 mM     84       33                                         Trichloroacetic acid                                                                          5% Sup.   11        4                                                        Ppt.      256       96                                         Amm. Sulfate    70% Sup.  11        2                                                        Ppt.      538       98                                         ______________________________________                                         Oncoinhibin samples were treated with various agents, dialyzed and then       assayed for biological activity.                                         

Oncoinhibin can be concentrated by trichloroacetic acid and ammoniumsulfate. Oncoinhibin was treated with different concentrations of eitherTCA or NH₄ SO₄, centrifuged, resuspended, dialyzed and then assayed forbiolobical activity. The results shown in Table IX indicate that all theactivity of Oncoinhibin can be precipitated by either 5% TCA or by 70%(SAS) ammonium sulfate. Thus, these results also suggest theproteinaceous nature of Oncoinhibin.

Amphiregulin is glycoprotein that was isolated from phorbolester-treated MCF-7 cells and inhibits the growth of A431 cells.Amphiregulin has an apparent molecular weight of 22.5 kDa. BothOncoinhibin and amphiregulin inhibit the growth of tumor cells.Oncoinhibin, however, is not produced by phorbol-ester treated MCF-7cells. Secondly, even though both Oncoinhibin and amphiregulin exhibitantiproliferative activity against A431 cells (Table VII), amphiregulin,in contrast to Oncoinhibin, is inactive against human melanoma (A375),human adenocarcinoma of the breast (ZR-75-1 or MCF-7), humanadenocarcinoma of the lung (A-549), human carcinoma of colon (H3347)human lymphoblastoid T cells (CEM), human EBV tranformed B cells, humanepidermal carcinoma of larynx (Hep 2), bovine fetal heart endothelialcells (CRL-1395), murine BALB/3T3, and minl lung (CCL-64) cells. Similarto Oncoinhibin, amphiregulin stimulates the proliferation of humanfibroblasts. Besides human fibroblasts, amphiregulin also stimulates thegrowth of certain tumor cells including human pituitary tumor cells (CRL7386), human ovarian carcinoma cells (HTB 77), African green monkeykidney cells (BSC-1) and rat kidney cells (NRK). The molecular weight ofOncoinhibin, however, is also significantly different from that ofamphiregulin and the mature form of the latter is an 84 amino acid longprotein.

Oncoinhibin is stable to both acidic and alkaline conditions at a pHrange of 2.0-10.0 (Table VIII). Several cytokines have been reportedwhich are stable to pH 2, including IFN-α, IFN-β, IL-2, IL-4, IL-8,CSF-1, GM-CSF, TGF-β, Oncostatin M and amphiregulin. Oncostatin M,amphiregulin, CSF-1 and IL-4 were also found stable to alkalineconditions (pH 12). Recently, lipolysis promoting factor (LPF), aprotein with an approximate molecualr weight of 6 kDa, has been isolatedfrom A375 melanoma cell line which is stable to heat 96° C. for 10minutes), protease K (10 μg/ml), trypsin, pronase, RNase, DNase andperiodate oxidation. LPF can be precipitted by trichloroacetic acid(10%) without any loss of biological activity. Oncoinhibin was found tobe stable to heat up to 80° C. for 30 minutes and only partial activitywas lost at 100° C. (Table IV). A trypsin 10% (w/w) treatment ofOncoinhibin for 24 hours at 37° C. lead to a partial loss of biologicalactivity. This is similar to that observed with CSF-1, GM-CSF and LT.The biological activity of Oncoinhibin was also found stable to 0.5%SDS.

Oncoinhibin is distinct from Tumor Killing Factor (TKF), a cytokineidentified from human macrophage-monocyte hydridoma in that TKF is abasic protein (pI 8-9.0), has an apparent molecular weight of 56 kDa bygel filtration and can be eluted from Conconavaline A-sepharose with0.4M α-methyl mannoside.

Oncostatin M is a cytokine isolated from U-937 cells treated withphorbol ester and inhibits the growth of human melanoma cell line A375in a thymidine incorporation assay. Unstimulated U-937 cells do notexpress the gene or secrete this activity. Phytohemagglutinin-activatedhuman peripheral blood T lymphocytes also express the gene and secretethis cytokine. Oncostatin M is a glycoprotein with a molecular weight of28 kDa by SDS-PAGE and 18 kDa by gel filtration. It synergized withTGF-β but not with interferons. Oncostatin M has been shown to inhibitthe proliferation of HTB 10 neuroblastoma cells, A-549 lung carcinomacells, as well as A375 and SKMEL-28 melanoma cells; it does not,however, inhibit the proliferation of L-929 cells. In contrast,Oncoinhibin is produced by K-562 cells (not by U-937 cells) both in thepresence as well as in the absence of phorbol ester and affects L-929cells. Moreover, in contrast to Oncoinhibin, oncostatin M is arelatively weak inhibitor of A375 cells (FIG. 14).

Transforming growth factor-β (TGF-β) is cytokine which is a homodimerwith a molecualr weight of 25 kDa on SDS-PAGE and inhibits the growth ofseveral cell types of epithelial and mesenchymal origin including humanvascular endothelial cells, T and B lymphocytes. A-549, MCF-7 cells arealso inhibited by TGF-β in an autocrine manner. However unlikeOncoinhibin, TGF-β is produced by a wide variety of cells includingplatelets, bone tissue and lymphocytes, and requires acid-activationbefore its activity can be examined. In addition, TGF-β has a molecularweight of 12.5 kDa on SDS-PAGE under reduced conditions.

Cytokine ELISA assays: A commercially available (R&D Systems)quantitative "sandwich" enzyme immunoassay technique was used to examinethe presence of known cytokines (TNF, LT, IL-1, IL-6 and IL-8) in theOncoinhibin preparation. The standard protocol provided by the supplierwas used. Briefly, a monoclonal antibody specific for differentcytokines was coated onto the microtiter plates and allowed to set upovernight to immobilize the antibodies. Then the samples were pipettedinto the wells and the cytokine if any is captured by the immobilizedantibody. After washing away any unbound sample proteins, anenzyme-linked polyclonal antibody specific for a given ctyokine wasadded to the wells and allowed to bind the cytokine which was boundduring the first incubation. Following a wash to remove any unboundpolyclonal antibody-enzyme reagent, a substrate solution was added tothe wells and color developed in proportion to the amount of cytokinebound in the initial step. Along with the samples tested, a series ofwells were prepared using known concentrations of the cytokinestandards. A curve plotting the optical density versus the concentrationof cytokine in these standard wells was prepared by comparing theoptical density of the samples to this standard curve. The concentrationof the cytokine in the unknown samples was then calculated (Table V,page 22).

Interleukin-1 is a cytokine produced by activated monocytes andfibroblasts, has a molecular weight of 17 kDa and inhibits the growth oftumor cell lines including ovarian carcinoma, A375 melanoma, K-562 andcertain breast tumor cell lines. Oncoinhibin, however, differs fromInterleukin-1 with respect to its source, molecular weight and tumorspecificity. Crude preparations of Oncoinhibin were examined for thepresence of IL-I by ELISA. The results shown in Table V demonstrate alack of presence of the IL-I protein in our Oncoinhibin preparation.

Interleukin-6 is cytokine produced by a wide variety of different celltypes in response to highly diverse stimuli and has a molecular weightof 26 kDa on SDS-PAGE. Specifically, IL-6 is also produced by normalhuman fibroblasts, U-937, human melanoma cell lines A375, RPMI-7951 etc.IL-6 inhibits the growth of myeloid leukemia and breast carcinoma celllines. Based on the source, method of induction and tumor cell targetspecificity, IL-6 appears to be a different cytokine from that ofOncoinhibin. Moreover, MCF-7 cells routinely used as a target forOncoinhibin are insensitive to the effect of Interleukin-6 (FIG. 16).Also in contrast to Oncoinhibin, IL-6 was found to inhibit the growth ofnormal human fibroblasts (FIG. 15).

Interferon-γ a cytokine with a molecular weight of 20-25 kDa onSDS-PAGE, is produced by T-lymphocytes when activated with variousmitogens and inhibits the growth of certain tumor cell lines. Thiscytokine is highly sensitive to acidic pH conditions. Oncoinhibindiffers from interferon-γ with respect to its source, method ofinduction and pH stability. Interferon-γ also differs from Oncoinhinbinwith respect to its effect on normal human fibroblasts. Oncoinhibinstimulates the proliferation whereas interferon-γ inhibits theTNF-induced fibroblast proliferation but not produced by Oncoinhibin(FIG. 18). Moreover, antibodies to interferon-γ do not reduce theactivity of Oncoinhibin but enhance it (FIG. 17).

Thus, the present invention provides a novel cytokine exhibiting diverseantineoplastic activity. The cytokine, Oncoinhibin, is secreted by humanerythroblastoid cells and has a molecular weight of approximately 28 kDaon SDS-PAGE. Production of Oncoinhibin appears to be enhanced in thepresence of phorbol ester. Oncoinhibin appears to be stable to a widerange of substances and stable in a wide pH range and to a hightemperature.

Due to its diverse neoplastic activity, it is contemplated thatOncoinhibin will be of therapeutic use in the treatment of a widevariety of neoplastic diseases, including carcinomas and lymphomas.Oncoinhibin may be supplied to humans or other animals as part of apharmaceutical composition that would contain a pharmaceuticallyacceptable carrier. Due to its diverse antineoplastic activity,Oncoinhibin will be useful in preventing recurrence of neoplasticdiseases. In addition, adminstration of Oncoinhibin to hosts having aneoplastic cells will likely extend the survival time of the host.Alternatively, neoplastic cells could be treated with Oncoinhibin invitro, e.g., the treatment and purging of bone marrow containingneoplastic cells. These methods of treating neoplastic cells asdescribed herein are well known in the art of cancer chemotherapy andconsequently a person having ordinary skill in this art could, withoutundue experimentation, determine the appropriate dosages and routes ofadministration of Oncoinhibin.

Oncoinhibin may also be useful as a novel immunomodulator. Oncoinhibinactivates lymphocytes, monocytes and neutrophils to kill tumor cells. Inaddition, Oncoinhibin may be therapeutically useful as a growth factor.Oncoinhibin stimulates the growth of normal cells.

In conclusion, it is seen that the present invention and the embodimentsdisclosed herein are well adapted to carry out the objectives and obtainthe end set forth in this application. Certain changes can be made inthe method and apparatus without parting from the spirit and scope ofthis invention. It is realized that changes are possible and that it isfurther intended that each element or step presided in any of the filingclaims is to be understood as to referring to all equivalent elements orsteps for accomplishing the essentially the same results insubstantially the same or equivalent manner. It is intended to cover theinvention broadly in whatever form its principles may be utilized. Thepresent invention, therefore, is well adapted to carry out the objectsand obtain the ends and advantages mentions, as well as others inherenttherein.

What is claimed is:
 1. A purified and isolated human Oncoinhibin proteinthat is secreted by erythroblastoid cells (K-562), having a molecularweight of 28 kDa on SDS-PAGE, stability at pH=2-10 and temperatures4°-100° C., and exhibiting anti-neoplastic activity against breast(MCF-7) and cervical (HeLa) carcinoma cell lines and has proliferatingeffects on normal human foreskin fibroblasts.
 2. A pharmaceuticalcomposition, comprising the human Oncoinhibin of claim 1 and apharmaceutically acceptable carrier.
 3. A method for preparing humanOncoinhibin of claim 1, comprising the steps of:a) incubating humanerythroblastoid cells (k-562) in a media, b) harvesting thecell-conditioned media, and c) isolating and purifying the humanOncoinhibin from the media.
 4. The method of claim 3, wherein the cellsare incubated in a serum-free medium.
 5. The method of claim 3, whereinthe cells are incubated in RPMP1640.
 6. The method of claim 3, furthercomprising enhancing the production of human Oncoinhibin in humanerythroblastoid cells with phorbol ester.
 7. The method of claim 3,wherein the human oncoinhibin is isolated and purified by:a)ultrafiltering the cell-conditioned media containing the humanOncoinhibin of claim 1, b) dialyzing the ultrafiltered media, c)performing DEAE Affigel blue chromatography, d) performing sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and reverse phasehigh performance liquid chromatography.